Freshwater harmful algal blooms of cyanobacteria or cyanoHABs are increasing in frequency and impact worldwide due to natural and anthropogenic flooding events. In the USA, all 50 states now regularly experience major cyanoHAB incidents, including Florida and Ohio that have declared states of emergency in recent years to address increased cyanotoxin loads in public waterways posing human health, environmental, and economic threats. While harmful cyanobacteria and their toxins are actively monitored, one of the most neurotoxic cyanotoxins, the organophosphate anatoxin-a(s), is not monitored. This water-soluble, UV-insensitive, and zwitterionic toxin is notoriously reactive, which has led to the ongoing challenges in its environmental monitoring. We recently discovered the genes encoding anatoxin-a(s) biosynthesis from the planktonic cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through a series of genomic, chemical, and biochemical experiments, we have nearly reconstituted the entire biosynthetic pathway from arginine to anatoxin-a(s) with recombinant enzymes. Moreover, we identified nearly complete anatoxin-a(s) transcripts from the nearshore Western Basin of Lake Erie at Toledo, Ohio, suggesting that residents in Toledo as well as other communities across the USA may experience anatoxin-a(s) exposure without their knowledge and that of regional and national monitoring agencies. Our discovery sets the stage for this 2-year, R21 application to apply our biosynthetic expertise to fill the gap in knowledge about the prevalence, significance, and impact of this critical toxin in the environment. We propose four specific aims to address our broad goals. First, we plan to complete the functional assignment of all anatoxin-a(s) biosynthesis enzymes. Second, we will broadly analyze metagenomic and metatranscriptomic fresh water supplies for anatoxin-a(s) genes and to identify the producing cyanobacteria. Third, we aim to develop a rapid, PCR screen specific for environmental anatoxin- a(s) gene detection. And fourth, we will evaluate the native phosphatase AnsH and commercial phosphatases as anatoxin-a(s) degradation enzymes with potential bioremediation applications.